Human parainfluenza virus type 3 (HPIV3) in an important respiratory tract pathogen capable of causing diseases such as pneumonia, bronchiolitus and croup in infants, young children, elderly and immunocompromised individuals. There is no effective vaccine or treatment currently available for HPIV3 infections. The goal of this application is to gain a more complete understanding of the mechanism through which this virus replicates its RNA genome by better understanding the cis-acting sequences and viral proteins involved in genome replication. More specifically, an HPIV3 minigenome reverse genetics system will be used to characterize/define important sequences present in the 3' end of the antigenomic RNA needed for viral replication (Specific Aim 1). Additionally, in Specific Aim 2 an infectious clone reverse genetics system will be used to generate small plaque HPIV3 mutants containing replication promoter mutations. From these small plaque mutants, large plaque revertants will be isolated and sequenced to determine if any of the large plaque revertants contain second-site reversion mutations mapping to protein coding sequences. Identification of protein changes that compensate for mutations in promoter regions will provide strong genetic evidence for a specific RNA-protein interaction involved in RNA replication and greatly aid in the understanding of the replication of HPIV3 and other non-segmented, negative sense RNA viruses. Additionally, mutations characterized in this work may prove useful for incorporation into an effective vaccine for this virus. [unreadable] [unreadable] [unreadable]